The transparent cornea is made up of an anterior stratified epithelium, a collagenous stromal matrix containing fibroblasts called keratocytes, Descemet's membrane and a single-layered endothelium. We have put forth the refracton hypothesis attempting to relate cornea to lens. The cornerstone of this hypothesis is that the exceptionally abundant intracellular proteins in the cornea share properties with the multifunctional crystallins of the lens. Our current corneal research addresses the molecular basis for corneal-specific gene expression, the roles of the abundant, intracellular corneal proteins, the global patterns of corneal gene expression, and stratification of the corneal epithelium. For corneal-specific gene expression, we are continuing to investigate the mouse aldehyde dehydrogenase 3 (ALDH3a1), rabbit ALDH1a1 and zebrafish gelsolin promoter in both transfection and transgenic mouse experiments. In FY2002 we showed that gelsolin, an actin binding protein, comprises approximately half of the water-soluble protein of the zebrafish corneal epithelium, making it a putative corneal crystallin in this species. We also showed by in situ hybridization that gelsolin is expressed at the blastula stage in zebrafish embryos and in the notocord before it becomes concentrated in the eye. Microinjection of human gelsolin, zebrafish gelsolin mRNA, and gelsolin-specific morpholino oligonucleotides have now established that zebrafish gelsolin is required for dorsalization during embryogenesis. Indeed, injection of gelsolin mRNA or human gelsolin protein dorsalized the developing embryo, often resulting in axis duplication. Thus, gelsolin, which is specialized for corneal expression in adult zebrafish, also modulates embryonic dorsal/ventral pattern formation in this species. In addition, we have cloned a second gelsolin from zebrafish in FY2003 which is expressed ubiquitously, is not concentrated in the cornea, and apparently does not influence dorsal/ventral patterning in zebrafish. It shares approximately 60% amino acid sequence identity with the corneal gelsolin. We have also produced recombinant zebrafish gelsolin for future, collaborative crystallography studies. Finally, in FY2003 we established that each eye of the surace fish, Anableps anableps,has two corneas (one submerged, one in air), each of which concentrates gelsolin in the epithelium. The surface corneal epithelia are multilayered and accumulate glycogen as well as gelsolin. In FY2003 we completed our studies showing that the corneal epithelial cells of Pax6+/- Small eye mice carrying two different mutations (SeyNeu or SeyDey) are poorly stratified and have reduced amounts of ALDH3a1, transketolase (TKT) and cytokeratin 12 (K12), all characteristic of mouse corneas. This indicates that high expression of these proteins in the corneal epithelium require proper amounts of Pax6 during development. The 4 kb ALDH3 promoter/CAT reporter gene, shown earlier to be expressed specifically in corneal epithelial cells of transgenic mice, was stimulated 10-fold in co-transfection experiments with Pax6 in Cos 7 cells. Surprisingly, Pax5a, a Pax6 isoform, repressed promoter activation in co-transfection experiments. Thus, Pax6/5a ratios, which we show change during corneal development, may have a role in corneal ALDH3 expression in vivo. Numerous expression plasmids have been constructed to study the molecular basis of mouse ALDH3a1 further. We also showed in FY2003 that corneal epithelial cell adhesion is reduced in the Small eye mice. This is associated with reductions in desmoglein, and in beta and gamma-catenins. We showed earlier that ALDH1a1 is the principal protein in rabbit corneal keratocyes and corneal epithelium. In FY2002 we showed corneal preference in rabbit ALDH1a1 promoter activity in transgenic mice. In FY2003 we established that XRE and an E-box cis-control elements contribute to the regulation of the rabbit ALDH1 gene in the cornea via hypoxia-related pathways. In FY2002 we reported that ALDH3a1 null mice show no abnormal phenotype, and that there is no evidence for the appearance of a compensatory protein in the cornea. In FY2003 we have provided preliminary evidence that the absence of ALDH3a1 may lead to increased apoptosis in the corneal epithelium. In FY2002 we showed that TKT null mice die in the early cleavage stages of development. However, the heterozygote TKT mice are viable and show no corneal abnormalities, although the mice were unexpectedly on average about 30% smaller than wild type mice with a proportionally smaller liver, testis, ovary and, especially, fat tissue. TKT heterozygote females gave small litters when mated with wild type males. These dose-dependent TKT phenotypes have clinical relevance. From a corneal perspective, it remains baffling that such profound reductions and eliminations of soluble proteins and metabolic enzymes have no affect of corneal morphology or clarity. In FY2003 we continued our efforts to produce a mouse line that contains a floxed TKT gene and a line that produces Cre in the corneal epithelial cells. These will allow numerous experiments, including the ability to knockout the TKT (and other) genes specifically in the developing cornea. In FY2002 we began to explore global gene expression in 6-week-old and 9-day-old mouse corneas by making and analyzing SAGE libraries. 60,000 tags, each representing the expression of a cDNA, were sequenced from each stage. In short, the pattern of genes expressed is characteristic of the tissue and is in good agreement with what is known about the expression of corneal proteins. Numerous genes expressed in these libraries are not represented in published SAGE libraries from other tissues (e.g. retina, lymphocytes, etc.). Many genes were identified that either increase or decrease in expression between these two stages of corneal development. In brief, our SAGE data demonstrate dynamic changes in gene expression after eye opening, and provide new probes for exploring corneal epithelial cell stratification, development and function, and for exploring the intricate relationship between programmed and environmentally induced gene epxression in the cornea. We anticipate that this fundamental data on corneal gene expression will have many applications in the future to biomedical problems of the cornea. Finally, in FY2003 we showed that mouse and bovine cornea have a similar content and spatial distribution of serum albumin, which is located in the stroma. The appreciable serum albumin in the cornea raises the possibility that it contributes to the physiological or optical functions of the cornea. Moreover, serum albumin's ability to bind drugs suggests that mice corneas could be exploited to study drug-serum albumin interactions in vivo and to test the usefulness of serum albumin as a drug carrier for corneal disorders.